Method for monitoring stability of polysaccharide-protein conjugate vaccines

Abstract

The present disclosure provides a process for assaying stability of monovalent and/or multivalent, liquid and lyophilized polysaccharide protein conjugate vaccine compositions using HPLC-SEC method. The method provides stability analysis (lot to lot) of polysaccharide protein conjugate vaccine with respect to aggregation profile, molar mass distribution and/or molecular size distribution, and data can be utilized for quality control during storage and batch release. The method is performed in the presence of multiple carrier proteins, free polysaccharides and excipient, without any interference of said components.

1 . A method for determining the stability of a polysaccharide-protein conjugate vaccine, said method comprising the following steps:
 a) subjecting the said polysaccharide-protein conjugate vaccine to a high performance size exclusion chromatography (HPLC-SEC) using a mobile phase and set of three chromatography columns in series to obtain an eluate;   b) passing the said eluate through detectors and evaluating the eluate to obtain the distribution of the molecules with respect to molecular weight; and   c) analysing the molecular weight to obtain molecular size and/or molar mass profile of the polysaccharide-protein conjugate vaccine based on the percentage of high molecular weight (HMW), average molecular weight (AMW) and low molecular weight (LMW) molecules.   
     
     
         2 . The method as claimed in  claim 1 , wherein a pre-treatment step is carried out before step a). 
     
     
         3 . The method as claimed in  claim 2 , wherein the pre-treatment step comprises of reconstitution of polysaccharide-protein conjugate vaccine using a buffer. 
     
     
         4 . The method as claimed in  claim 3 , wherein the buffer used for reconstitution of polysaccharide-protein conjugate vaccine in pre-treatment step is selected from phosphate buffer saline, Tris, MES, HEPES, citrate and combinations thereof. 
     
     
         5 . The method as claimed in  claim 4 , wherein the buffer used for reconstitution of polysaccharide-protein conjugate vaccine in pre-treatment step is Tris buffer. 
     
     
         6 . The method as claimed in  claim 1 , wherein the set of three chromatography columns in series is connected with a guard column. 
     
     
         7 . The method as claimed in  claim 6 , wherein the arrangement of columns is guard column followed by first column, first column followed by second column and second column followed by third column. 
     
     
         8 . The method as claimed in  claim 6 , wherein the guard column comprises polymer based packed material having particle size 9-14 µm. 
     
     
         9 . The method as claimed in  claim 8 , wherein the polymer based packed material of guard column comprises polyhydroxymethacrylate based material. 
     
     
         10 . The method as claimed in  claim 7 , wherein the first and second chromatography columns comprises polyhydroxymethacrylate based material and third chromatography column is selected from hydroxylated polymethacrylate based material and hydrophilic vinyl polymer based material. 
     
     
         11 . The method as claimed in  claim 7 , wherein particle size of first column is 34 - 36 µm, particle size of second column is 12 - 14 µm and particle size of third column is 9 - 14 µm. 
     
     
         12 . The method as claimed in  claim 1 , wherein the length of the set of three chromatography columns in series ranges from 85 - 95 cm. 
     
     
         13 . The method as claimed in  claim 6 , wherein the guard column and chromatography columns in series are connected using a connector. 
     
     
         14 . The method as claimed in  claim 1 , wherein the detector in step b) is selected from UV detector, refractive index (RI) detector and multiangle light scattering (MALS) detector and combinations thereof. 
     
     
         15 . The method as claimed in  claim 1 , wherein the mobile phase is a buffer selected from phosphate buffer saline, Tris, MES, HEPES, citrate and combinations thereof. 
     
     
         16 . The method as claimed in  claim 15 , wherein the buffer is phosphate buffer saline having pH ranging from 7.2 to 7.5, preferably pH 7.4. 
     
     
         17 . The method as claimed in  claim 16 , wherein the phosphate buffer saline comprises of sodium chloride in the range of 20 to 40 g, preferably 23.38 g. 
     
     
         18 . The method as claimed in  claim 1 , wherein the high performance size exclusion chromatography (HPLC-SEC) in step a) comprises a flow rate ranging from 0.1 to 1 ml per minute, preferably 0.30 to 0.80 ml per minute. 
     
     
         19 . The method as claimed in  claim 1 , wherein the high performance size exclusion chromatography (HPLC-SEC) in step a) comprises a column temperature ranging from 25° C. to 35° C. 
     
     
         20 . The method as claimed in  claim 1 , wherein the high performance size exclusion chromatography (HPLC-SEC) in step a) comprises injection run time ranging from 60 - 70 min. 
     
     
         21 . The method as claimed in  claim 1 , wherein the polysaccharide-protein conjugate vaccine is in liquid form or lyophilized form. 
     
     
         22 . The method as claimed in  claim 1 , wherein the molecular weight is obtained in the form of high molecular weight, average molecular weight and low molecular weight. 
     
     
         23 . The method as claimed in  claim 1 , wherein the polysaccharide is a bacterial capsular polysaccharide, and is obtained from group comprising of  Helicobacter   pylori ,  Chlamydia   pneumoniae,   Chlamydia   trachomatis,   Ureaplasma   urealyticum,   Mycoplasma   pneumoniae,   Staphylococcus   spp.,   Staphylococcus   aureus,   Streptococcus   spp. , Group A  Streptococcus , Group B  Streptococcus,   Streptococcus   agalactiae,   Streptococcus   pyogenes,   Streptococcus   pneumoniae,   Streptococcus   viridans,   Enterococcus   faecalis,   Neisseria   meningitidis,   Neisseria   gonorrhoeae,   Bacillus   anthracis,   Salmonella   spp.,   Salmonella   typhi,   Salmonella   paratyphi,   Salmonella   enteritidis,   Salmonella   typhimurium,   Vibrio   cholerae,   Pasteurella   pestis,   Pseudomonas   aeruginosa,   Campylobacter   spp.,   Campylobacter   jejuni,   Clostridium   spp.,   Clostridium   difficile,   Mycobacterium   spp.,   Mycobacterium   tuberculosis,   Treponema   spp.,   Borrelia   spp.,   Borrelia   burgdorferi,   Leptospira   spp.,   Hemophilus   ducreyi,   Corynebacterium   diphtheria,   Bordetella   pertussis,   Bordetella   parapertussis,   Bordetella   bronchiseptica,   Hemophilus   influenzae,   Escherichia   coli,   Shigella   spp.,   Ehrlichia   spp.,   and   Rickettsia   spp. . 
     
     
         24 . The method as claimed in  claim 23 , wherein polysaccharide is obtained from one or more of the following:
 a.  Streptococcus   pneumoniae  serotype 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11 A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48;   b.  Neisseria   meningitidis  serotypes A, B, C, D, W135, X, Y, Z, and 29E;   c.  Haemophilus   influenzae  type b;   d.  Salmonella   spp.  including  salmonella   typhi,   salmonella   paratyphi  A,  salmonella   paratyphi  B,  salmonella   paratyphi  C,  salmonella   typhimurium , and  salmonella   enteritidis ;   e.  Streptococcus   spp.  including Group A  Streptococcus  and Group B  Streptococcus ,  Streptococcus  group (Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX).   
     
     
         25 . The method as claimed in  claim 1 , wherein the polysaccharide-protein conjugate vaccine to be evaluated comprises carrier protein selected from the group of CRM197, diphtheria toxin/toxoid,  Neisseria   meningitidis  outer membrane complex, fragment C of tetanus toxoid, tetanus toxin/toxoid, pertussis toxin/toxoid, flagellin (FliC), cholera toxin B subunit (CTB), protein D of  H.   influenzae ,  E.   coli  LT,  E.   coli  ST, and exotoxin A from  Pseudomonas   aeruginosa , outer membrane complex c (OMPC), porins (Por A, Por B), transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), PhtA, PhtB, PhtE, pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11,  M.   catarrhalis  uspA, protective antigen (PA) of  Bacillus   anthracis  and detoxified edema factor (EF) and lethal factor (LF) of  Bacillus   anthracis , ovalbumin, keyhole limpet hemocyanin (KLH), C5a peptidase group A or group B  Streptococcus , human serum albumin, bovine serum albumin (BSA), NTHi high molecular weight protein, fHbp, purified protein derivative of tuberculin (PPD) and Cholera toxin subunit B. 
     
     
         26 . The method as claimed in  claim 24 , wherein the polysaccharide is selected from  Neisseria   meningitidis  serotypes A, C, Y, W135 and X. 
     
     
         27 . The method as claimed in  claim 25 , wherein the carrier protein is selected CRM197, tetanus toxoid (TT) and combinations thereof. 
     
     
         28 . The method as claimed in  claim 1 , wherein the polysaccharide-protein conjugate vaccine is a multivalent vaccine. 
     
     
         29 . The method as claimed in  claim 1 , wherein the polysaccharide-protein conjugate vaccine is a monovalent vaccine. 
     
     
         30 . The method as claimed in  claim 28 , wherein the multivalent vaccine comprises of  Neisseria   meningitidis  serotype A - TT conjugate,  Neisseria   meningitidis  serotype X
 TT conjugate,  Neisseria   meningitidis  serotype C - CRM197 conjugate,  Neisseria   meningitidis  serotype Y - CRM197 conjugate and  Neisseria   meningitidis  serotype W 
 CRM197 conjugate. 
 
     
     
         31 . The method as claimed in  claim 1 , wherein the high molecular weight is in the range from 13000 kDa to 19000 kDa, average molecular weight is in the range of 2000 kDa to 11000 kDa and low molecular weight is in the range of 200 kDa to 2000 kDa for lyophilized polysaccharide-protein conjugate vaccine. 
     
     
         32 . The method as claimed in  claim 1 , wherein the high molecular weight is in the range from 10000 kDa to 19000 kDa, average molecular weight is in the range of 1000 kDa to 10000 kDa and low molecular weight is in the range of 200 kDa to 1000 kDa for liquid polysaccharide-protein conjugate vaccine.